Ribonucleotide reductase is responsible for regulating a balanced and continued supply of deoxyribonucleotide precursors required for DNA replication. As an approach to understanding the structural and functional basis for regulation of ribonucleotide reduction, we have isolated and characterized a multi-enzyme containing ribonucleotide reductase proteins B1, B2 and thioredoxin. This form of ribonucleotide reductase is characterized as a highly active, and physically stable complex. Unlike the solubilized form of reductase which shows dilution inactivation, the specific activity in the membrane fraction is independent of protein concentration. The functional significance and the integrity of this membrane reductase complex is also suggested by co-purification of several other enzymes involved in deoxynucleotide metabolism, including thymidylate synthetase and DNA polymerase I. Present studies will continue characterization of the various enzyme activities copurifying with the ribonucleotide reductase-membrane fraction together with studies on any functional relations that exist between these proteins. The enzymatic behavior of the membrane associated form of reductase will be carried out using the crude membrane fraction, as well as the B1 protein purified as a lipoprotein complex on dATP-Sepharose. Thioredoxin, the hydrogen donor implicated in ribonucleotide reduction has been isolated as a phosphoprotein. The site of phosphorylation is one of the two cysteines previously implicated in hydrogen transfer. When isolated from late log cultures, thioredoxin is 86% in the phosphorylated form. Current studies seek to define the role of phosphothioredoxin in the hydrogen transfer reaction with ribonucleotide reductase.